CN107232184A - The dilution for preserving semen under normal temperature of Guanzhong Black Pig - Google Patents
The dilution for preserving semen under normal temperature of Guanzhong Black Pig Download PDFInfo
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- CN107232184A CN107232184A CN201710619760.4A CN201710619760A CN107232184A CN 107232184 A CN107232184 A CN 107232184A CN 201710619760 A CN201710619760 A CN 201710619760A CN 107232184 A CN107232184 A CN 107232184A
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- dilution
- normal temperature
- guanzhong
- black pig
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention discloses a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig, its main component is glucose, sodium citrate, ethylenediamine tetra-acetic acid, sodium acid carbonate, potassium chloride, kojic acid, and ε polylysines etc..The present invention can normal temperature preserves 1~5 day and reaches artificial insemination requirement in liquid form by Guanzhong Black Pig seminal fluid.Kojic acid is added in the dilution has good anti-oxidation protection effect to sperm; reduce usage amount of the antibiotic in Swine Production simultaneously; bacterial resistance sex chromosome mosaicism can effectively be alleviated; so that the pig semen for solving technology of artificial insemination during Guanzhong Black Pig large-scale cultivation preserves problem; public swine rearing is reduced, the utilization rate of excellent boar semen is improved.The dilution liquor manufacture method is simple, and simple to operate, production cost is low, is easy to large-scale promotion application.
Description
Technical field
The present invention relates to biological technical field, the dilution for preserving semen under normal temperature of more particularly to Guanzhong Black Pig deep semen deposition is matched somebody with somebody
Side.
Background technology
Guanzhong Black Pig is the first batch of geography symbol product kind of the Ministry of Agriculture, because producing and be distributed widely in Guanzhong, Shanxi Plain and
Gain the name.Guanzhong Black Pig be with Berkshire, Gansu Jingchuan BaMei swine, Sichuan Neijiang Pig selection cross meat dual-purpose type product
Kind, complete to identify the beginning of the eighties, with elite germplasm features such as reproductive capacity height, good meat, resistance to thick feeding, strong stress resistances.
During the large-scale cultivation of Guanzhong Black Pig is promoted, excellent herd boar can be improved using deep insemination technique
The service efficiency of seminal fluid, while reducing the number of animals raised of boar, reduces production cost, improves pig farm economic benefit.But at this
During the use of technology of artificial insemination, however it remains some more distinct issues.Such as, pig semen is in collection, dilution point
Be filled with and the later stage preserve during inevitably microbial contamination, due to microorganism have to the preservation of seminal fluid it is great
Harm, it is main at present by adding the growth that antibiotic suppresses bacterium in seminal fluid in dilution, to reduce its danger to sperm
Evil.But in recent years because the not scientifical use of antibiotic causes the generation of bacterial drug resistance, if continuing to add in dilution
Antibiotic may cause the infection sow that drug tolerant bacteria is oriented by seminal fluid, on the one hand when using the essence by these germ contaminations
The breeding achievement of sow may be caused after liquid semen deposition to be reduced, and on the other hand may be increased when these bacteriums trigger disease again
The treatment cost of sow.In the commercial operations of current pig semen, Boar spermatozoa during storage and transport due to film in contain
There is substantial amounts of unrighted acid and be highly prone to oxidative stress, this structural integrity and function to sperm, which are normally played, to be had extremely
Detrimental effect, the breeding achievement of sow after reduction semen deposition.Therefore in order to adapt to the development of Artificial Insemination Technology of Swine, promote the Central Shanxi Plain
Promoting the use of for black pig deep insemination technique, improves semen quality and sow breeding achievement that porcine semen at normal temperature is preserved, reduces pig
Field production cost, raising productivity effect, it is a kind of suitable for Guanzhong Black Pig semen at normal temperature in the urgent need to developing under existing situation
The dilution of preservation.
Commodity dilution powder brand is more, the good and bad jumbled together, there is problems with actual applications：
1) widely used porcine semen at normal temperature preservation dilution powder is mainly foreign brand name, domestic independent research in the market
Product it is less, and these dilution powders be not necessarily suitable Guanzhong Black Pig seminal fluid dilution preserve.
2) main addition antibiotic bactericidal in commodity dilution powder, but the residual of antibiotic and bacterium in recent years is resistance to
Property of medicine problem becomes increasingly conspicuous, and assistant officer is to be solved.
3) sperm easily produces oxidative stress phenomenon during normal temperature preservation, semen quality is influenceed, so as to reduce sow
Breeding achievement.
The content of the invention
It is an object of the invention to provide a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig.
To reach above-mentioned purpose, present invention employs following technical scheme.
The composition that the normal temperature preserves dilution includes distilled water, 35~40g/L glucose, 5.5~6.5g/L lemon
Sour sodium, 5.5~6.5g/L ethylenediamine tetra-acetic acid, 1.0~1.25g/L sodium acid carbonate, 0.5~1.0g/L potassium chloride,
0.02~0.06g/L kojic acid, 0.04~0.16g/L epsilon-polylysine.
It is preferred that, the normal temperature preserves dilution regulation osmotic pressure in 240mosm~380mosm, and pH is 6.4~7.2.
It is preferred that, the composition that the normal temperature preserves dilution includes distilled water, 37.15g/L glucose, 6.0g/L lemon
Lemon acid sodium, 6.0g/L ethylenediamine tetra-acetic acid, 1.25g/L sodium acid carbonate, 0.75g/L potassium chloride, 0.02~0.06g/L
The epsilon-polylysine of kojic acid and 0.04~0.16g/L.Kojic acid, epsilon-polylysine consumption and the motility rate, the acrosome that preserve sperm are complete
Whole property, membrane integrity are related.
It is further preferred that the normal temperature preserve dilution composition include distilled water, 37.15g/L glucose,
6.0g/L sodium citrate, 6.0g/L ethylenediamine tetra-acetic acid, 1.25g/L sodium acid carbonate, 0.75g/L potassium chloride,
0.04g/L kojic acid and 0.08g/L epsilon-polylysine.
It is preferred that, after the normal temperature preservation dilution is mixed with seminal fluid, the density of sperm is diluted to 2 × 107~3 × 107
Individual/mL, the holding time is at least 120 hours.
It is preferred that, the normal temperature preserves dilution and mixed with seminal fluid after being preserved (optimal save strategy temperature at 15~20 DEG C
Spend for 17 DEG C).
Beneficial effects of the present invention are embodied in：
The dilution for preserving semen under normal temperature of Guanzhong Black Pig of the present invention is using the Semen routine problem of Guanzhong Black Pig to set out
Point, composition develops the dilution that the efficient normal temperature of energy preserves seminal fluid based on glucose-sodium citrate-sodium acid carbonate-potassium chloride
Formula of liquid.Dilution of the present invention has good normal temperature liquid storage effect to Guanzhong Black Pig seminal fluid, solves Guanzhong Black
The pig semen of technology of artificial insemination preserves problem during pig large-scale cultivation, reduces public swine rearing, improves excellent public affairs
The utilization rate of pig semen；The dilution liquor manufacture method is simple simultaneously, and production cost is low, easily uses, and is easy to large-scale promotion should
With.
Embodiment
The present invention is elaborated with reference to embodiment.
(1) the dilution for preserving semen under normal temperature formula in the semen deposition of Guanzhong Black Pig deep
The present invention is using the Semen routine problem in the semen deposition of Guanzhong Black Pig deep as starting point, with glucose-sodium citrate-carbon
Composition based on sour hydrogen sodium-potassium chloride, addition cationic antibacterial peptide epsilon-polylysine and antioxidant kojic acid are developed can be efficiently
Addition kojic acid has good anti-oxidant guarantor to sperm in the dilution formula of liquid of (15~20 DEG C) preservation seminal fluid of normal temperature, the dilution
Effect and certain antibacterial ability are protected, further combined with cationic antibacterial peptide epsilon-polylysine substitute antibiotics are used, both effectively
The easy oxidative stress problem of sperm during porcine semen at normal temperature is preserved is alleviated, antibiotic making in Swine Production is reduced again
Consumption, can effectively alleviate bacterial resistance sex chromosome mosaicism, and the use and drug-resistant bacteria for greatly reducing antibiotic are produced.The present invention has
Beneficial to the seminal fluid utilization rate for expanding excellent herd boar, ensure sow health and improve the conception rate of deep semen deposition, realize efficiently low
Honest and clean deep semen deposition.
The dilution for preserving semen under normal temperature of Guanzhong Black Pig deep disclosed by the invention semen deposition, its formula is in glucose, lemon
Lemon acid sodium, ethylenediamine tetra-acetic acid, sodium acid carbonate, 0.02~0.06g/L of addition kojic acid and 0.04 on the basis of potassium chloride~
0.16g/L epsilon-polylysine, is mixed with distilled water, and regulation osmotic pressure is in 240mosm~380mosm, and pH is 6.4~7.2, i.e.,
Form normal temperature and preserve dilution.
Glucose, which has, provides nutrition to supplement sperm existence and the consumed energy of motion, maintains the work of seminal fluid osmotic pressure
With.Sodium citrate and sodium acid carbonate have pressed in non-toxic, pH modulabilities and good buffering stability, dilution of the present invention than
The sodium citrate and sodium acid carbonate of example addition have reached highly desirable offer buffering and equilibrium osmotic pressure action effect.Ethylenediamine tetraacetic
Acetic acid is a kind of nontoxic organic sequestering agent, can reduce damaging action of the metal ion to sperm with metal ion-chelant.Chlorine
Change the addition of potassium for maintaining the normal operation of spermatoblast sodium potassium pump to play an important roll.The invention is characterized in that：1. do not add
Added with antibiotic, not only can effectively suppress the propagation of bacterium in seminal fluid, ensure the external storage of sperm, also reduce herding life
Antibiotic using and remains in production, reduces the probability of drug tolerant bacteria generation.2. there is extremely strong inoxidizability, suppress thin
The characteristics such as bacteria growing, chelated metal ions, the lipid peroxidation that can effectively reduce harmful substance active oxygen radical (ROS) is anti-
Should, improve sperm TAC (T-AOC).Therefore, with raising sperm motility rate and exercise performance, membrane integrity, top
The effects such as body integrality and mitochondrial integrity.
The semen deposition of above-mentioned Guanzhong Black Pig deep dilution for preserving semen under normal temperature formula another feature is that it is simple to operate and
It is economical and practical, it is with fresh smart isothermal dilution, sealing preserve after each components in certain proportion is fully mixed, seminal fluid is favourable with air exclusion
In damage of the sperm from external environment.Motility rate still can reach more than 60% after depositing 120 hours at normal temperatures, fully meet kind
The demand that pig farm is inseminated deep.
The dilution for preserving semen under normal temperature of Guanzhong Black Pig deep semen deposition, its composition optimum content is：37.15g glucose,
6.0g sodium citrate, 6.0g ethylenediamine tetra-acetic acid, 1.25g sodium acid carbonate, 0.75g potassium chloride, 0.04g kojic acid,
0.08g epsilon-polylysine.The seminal fluid of Guanzhong Black Pig deep semen deposition is obtained after mentioned component and 1L distilled waters are fully mixed
Normal temperature preserves dilution.Dilution pH value is 6.4~6.8.
(2) the fresh smart store method of Guanzhong Black Pig
Fresh wonderful collection and performance rating：Adult healthy herd boar seminal fluid is gathered using double gloves hand grip, at 37 DEG C
Motility rate and density to sperm carry out performance rating, and the motility rate and fortune of sperm are detected under computer-aided analysis system (CASA)
Dynamic performance, sample of the motility rate higher than 80% can be diluted and preserve, and seminal fluid density is in computer-aided analysis system
(CASA) detecting can detect and show simultaneously during sperm motility rate.
Normal temperature store method：According to adopted fresh essence (temperature is 37 DEG C) volume and density, the Guanzhong Black of isothermal is added
The dilution for preserving semen under normal temperature (under optimum formula content) of pig deep semen deposition, it is 2.5 × 10 to make sperm concentration7Individual/mL, gently
Rock and be divided in after mixing in 80mL seminal fluid bottle, according to temperature after being transferred to 17 DEG C of perseverances after 1~2h of seminal fluid slow cooling to room temperature
Temperature refrigerator preserves and is 120h, during which can need the deep semen deposition for pig according to production.
(3) performance rating after Guanzhong Black Pig Semen routine
Sperm motility rate is determined：10 μ L samples are taken to detect sperm motility rate under computer-aided analysis system (CASA) before semen deposition
And exercise performance.Sperm motility performance parameter mainly includes sperm linear motion speed, curvilinear motion speed and sperm side-sway width
Degree.
Plasmalemmae of sperms integrity detection：Detected using hypo-osmotic swelling test (HOST) method.With fructose-sodium citrate hypotonic medium
Sperm concentration is adjusted to 2 × 106Individual sperm/mL, 37 DEG C of incubation 30min, takes 10 μ L seminal fluid to be engraved under 400 times of phase contrast microscopes
Sperm swelling situation is calculated, plasmalemmae of sperms percentage of head rice is counted.
The detection of Sperm acrasomal integvity：Take 10 μ L samples to make smear, use fluorescent dye fluorescein isothiocynate-flower
After raw agglutinin (FITC-PNA) combined staining, every 200 Sperm acrasomal integvities are calculated at random under fluorescence microscope, calculate
More than 3 times.
The detection of mitochondria of sperms film integrality：Take 10 μ L samples to make smear, use fluorescent dyes rhodamine-iodate third
After pyridine (Rh123-PI) combined staining, every 200 mitochondria of sperms film percentage of head rice is calculated at random under fluorescence microscope, calculate 3
More than secondary.
(4) specific experiment example
3 adult healthy Guanzhong Black Pig herd boar (1.2~2 one full year of life) seminal fluid are gathered using hand grip, computer aided manufacturing is used
Analysis system (CASA) is helped to detect the motility rate and density of seminal fluid.Using above-mentioned dilution and operating method by Semen routine
To the explanation time, experiment contrast is used as using traditional BTS liquid.Performance rating is carried out to every boar semen.As a result show to be gathered
The fresh smart sample rate of herd boar is 6.2~12.5 × 109Change in the range of individual/mL, averag density is 0.89, and motility rate exists
Change in the range of 82.69%~91.78%, average value is 86.75%.Normal temperature preserve after 120h motility rate 65.58%~
Change in the range of 67.26%, average value is 66.42%, acrosomal integrity average value is 79.59%, plasm membrane integrity average value
For 56.77%.Data result is shown in Table 1.
The effect of dilution of the present invention can be mainly sperm extra-nutrition material, maintain the osmotic pressure of seminal fluid, reduce essence
Active oxygen is to sperm membrane knot in harm of the bacterium to sperm in liquid, harm of the reduction sperm metabolism product to sperm, reduction seminal fluid
The harm of structure.Prove that dilution formula of liquid disclosed by the invention is preserved with good effect to the normal temperature of Guanzhong Black Pig seminal fluid by experiment
(for example, 0.08g/L epsilon-polylysines and 0.04g/L kojic acid do not interfere with the osmotic pressure of seminal fluid, effectively it can really control in seminal fluid
Bacterial number；Normal temperature preserves smart sperm motility rate after 120h and meets production needs up to more than 60%) effectively it can suppress in seminal fluid
The growth of bacterium, keep sperm motility rate, acrosomal integrity, reduction rate of teratosperm, swine artificial insemination conception rate can be effectively improved
And inseminatio externalis efficiency, improve artificial insemination production efficiency.Kojic acid is safe and harmless with epsilon-polylysine in dilution simultaneously
, the safe material of drug residue free.
Influence of the dilution of the present invention of table 1. to Guanzhong Black Pig Semen liquid storage
In addition, the present invention has anti-oxidant and two kinds of effects of antibacterial simultaneously, kojic acid, two kinds of materials of epsilon-polylysine are all in food
Applied in product addition, it is nuisanceless and cost is relatively low, and the use of antibiotic in production can be reduced.
Claims (7)
1. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig, it is characterised in that：The composition that the normal temperature preserves dilution includes
Distilled water, 35~40g/L glucose, 5.5~6.5g/L sodium citrate, 5.5~6.5g/L ethylenediamine tetra-acetic acid, 1.0
The kojic acid of~1.25g/L sodium acid carbonate, 0.5~1.0g/L potassium chloride and 0.02~0.06g/L.
2. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig according to claim 1, it is characterised in that：The normal temperature is protected
Depositing the composition of dilution also includes 0.04~0.16g/L epsilon-polylysine.
3. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig according to claim 1 or claim 2, it is characterised in that：It is described normal
It is 240mosm~380mosm that temperature, which preserves dilution osmotic pressure, and pH is 6.4~7.2.
4. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig according to claim 1 or claim 2, it is characterised in that：It is described normal
The composition that temperature preserves dilution includes distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L ethylenediamine
Tetraacethyl, 1.25g/L sodium acid carbonate, 0.75g/L potassium chloride, 0.02~0.06g/L kojic acid and 0.04~0.16g/L
Epsilon-polylysine.
5. according to a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig of claim 1 or 2 or described, it is characterised in that：It is described
The composition that normal temperature preserves dilution includes distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L second two
Amine tetraacethyl, 1.25g/L sodium acid carbonate, 0.75g/L potassium chloride, 0.04g/L kojic acid and 0.08g/L ε-poly- bad ammonia
Acid.
6. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig according to claim 1 or claim 2, it is characterised in that：It is described normal
After temperature preservation dilution is mixed with seminal fluid, the density of sperm is diluted to 2 × 107~3 × 107Individual/mL, the holding time is at least 120
Hour.
7. a kind of dilution for preserving semen under normal temperature of Guanzhong Black Pig according to claim 1 or claim 2, it is characterised in that：It is described normal
Temperature preservation dilution is mixed with seminal fluid to be preserved after at 15~20 DEG C.
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| CN201710619760.4A CN107232184B (en) | 2017-07-26 | 2017-07-26 | Diluent for preserving semen of black pig in Guanzhong at normal temperature |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110326612A (en) * | 2019-08-21 | 2019-10-15 | 西北农林科技大学 | Anti-oxidant dilution of a kind of porcine semen at normal temperature preservation and preparation method thereof |
| CN113317312A (en) * | 2021-06-01 | 2021-08-31 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
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| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
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| CN1712519A (en) * | 2004-06-25 | 2005-12-28 | 华中农业大学 | Liquid for preserving pig semen |
| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110326612A (en) * | 2019-08-21 | 2019-10-15 | 西北农林科技大学 | Anti-oxidant dilution of a kind of porcine semen at normal temperature preservation and preparation method thereof |
| CN113317312A (en) * | 2021-06-01 | 2021-08-31 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
| CN113317312B (en) * | 2021-06-01 | 2022-07-12 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
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